title: noninfected_m464_b1
description: Protocols: RNA was extracted (see nucleic acid extraction protocol) from small intestinal epithelium (see below) from mice with citrobacter infection (see treatment protocol) and not, a library was created (see nucleic acid library construction protocol) and sequenced paired end 150 bp (see nucleic acid sequencing protocol)Crypts from distal colon were isolated according to a slight modification of a published protocol (Sato 2013). In summary, the distal colon of mouse was washed in ice-cold PBS and opened longitudinally. A microscopy cover slip was used to gently scrape of excess mucus before the colon was cut into 2-4 mm pieces. The fragments were resuspended in 30 mL ice-cold PBS and pipetted up and down with a 10 mL pipette. Supernatant was discarded and this step was repeated 5-10 times until the supernatant was clear. The fragments were incubated in 10 mM EDTA in PBS at 4 C for 30 min with gentle rocking. The supernatant was removed, 20 mL of ice-cold PBS was added and the fragments were pipetted up and down. This washing step was repeated until the crypt fraction appeared as seen in a microscope. The 1-3 consecutive crypt fractions were passed through a 70 um cell strainer and collected into a FCS coated 50 mL tube. The crypts were spun down at 300 xg for 5 min and washed once in 10 mL ice cold PBS at 200 xg for 5 min. References:Sato, Toshiro and Hans Clevers (2013). “Primary Mouse Small Intestinal Epithelial Cell Cultures”. In:Epithelial Cell Culture Protocols: Second Edition. Ed. by Scott H. Randell and M. Leslie Fulcher. Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 319-328.isbn: 978-1-62703-125-7.doi:10.1007/978-1-62703-125-7_19.527url:https://doi.org/10.1007/978-1-62703-125-7_19. The mice studies for Citrobacter rodentium infection were approved by Norwegian Food Safety Authority (FOTS ID: 11842). Briefly, C. rodentium was grown at 37 C in Luria- Bertani (LB) medium supplemented with chloramphenicol (30 ug/ml) overnight. 6-8 weeks old C57BL/6 mice were orally gavaged with a culture of 10e8 - 10e9 CFU per mouse delivered in a volume of 100 uL of sterile PBS. Mice remained infected with C. rodentium for six days. To determine the infection level, fecal samples were collected on day 5, homogenized in sterile PBS with a FastPrep homogenizer. After serial dilution of fecal samples, homogenates were plated on chloramphenicol resistant agar plates and the plates were counted after 24 h of growth at 37 C. After 6 days of infection, mice were euthanized by CO2 inhalation, parts of distal colon tissue w RNA was extracted with Quick-RNA Microprep Kit according to the manufacturers instructions (Zymo, R1050). Library preparation was done with NEB Next(R) Ultra(TM) RNA Library Prep Kit
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-05-01T00:23:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-03T20:36:11Z
attributes.tag: External Id
attributes.value: SAMEA6872472
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:35Z
attributes.tag: INSDC last update
attributes.value: 2020-06-03T20:36:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9184:noninfected_m464_b1
attributes.tag: age
attributes.value: 72
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: J
attributes.tag: organism part
attributes.value: crypt of Lieberkuhn of large intestine
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-9184:noninfected_m464_b1
attributes.tag: sampling site
attributes.value: distal colon
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6